Our long-term goal is to better understand the etiologic roles of protein posttranslational modifications (PTMs). This project focuses on isoaspartic acid (isoAsp, isoD or beta-Asp), which is generated spontaneously from either asparagine deamidation or aspartic acid isomerization. IsoAsp has been found in myriad proteins (e.g., beta-amyloid in the brain and crystalline in the eyes). Notably, isoAsp imparts a D-configuration and beta-linkage into the peptide backbone; such alteration of peptide backbones is exceptionally rare and may drastically alter protein structure and function. While well-recognized in aging, its emerging roles in signaling, regulation and autoimmunity have yet been widely appreciated. A lack of comprehensive knowledge of isoAsp and the unique technical challenges in its analysis hamper progress in this field. For one thing, isoAsp formation is the smallest PTM by mass change (Da; isoAsp has the same mass as Asp). Recently, these technical barriers have been overcome by novel approaches that combine chemo-enzymatic labeling and enrichment with mass spectrometry. Four specific aims will be undertaken. First, proteomic analysis of isoAsp in samples with biochemical and medical relevance will be performed. Second, tight binders to isoAsp will be engineered and used as antibodies for applications that complement existing methodologies. Third, the substrate specificity of the repair enzyme protein isoaspartate methyltransferase (PIMT) will be probed, focusing most assiduously on poor substrates of PIMT. Isoaspartyl peptide library screening, enzyme kinetic characterization, proteomic analysis and protein structural modeling will be integrated to augur the fate of isoAsp in individual proteins and whole systems. Last, alternative metabolic pathways for isoAsp will be discovered. The proposed research will lead to the discovery of biomarkers of human diseases, and effective methods of prevention, intervention and treatment for age-related conditions, neurodegenerative disorders (e.g., Alzheimer's disease), autoimmune diseases and other isoAsp-related ailments. PUBLIC HEALTH RELEVANCE: Isoaspartic acid formation is one of the most common, yet under-appreciated, modifications of proteins, and may significantly alter protein structures and functions. A comprehensive knowledge of this ubiquitous protein modification will advance both our understanding of the etiological mechanism and the intervention of diseases, particularly neurodegenerative and age-related disorders.